Differences in subtype distribution between screen-detected and symptomatic invasive breast cancer and their impact on survival.

PURPOSE: Stage shift is considered a major reason for more favorable outcomes in patients with screen-detected breast cancer. However, even after adjusting for clinical stage, unresolved issues concerning the reasons for a survival benefit associated with screening programs remain. This study aims to evaluate differences in subtype distribution and outcomes among patients with screen-detected and symptomatic invasive breast cancer and assess whether variations in subtype distribution could explain differences in prognosis. METHODS: Survival analysis was performed to estimate the likelihood of distant recurrence and death in 1132 patients. Subtypes were defined as luminal A [estrogen receptor (ER)+ and/or progesterone receptor (PR)+, human epidermal growth factor receptor 2 (HER2)-, and Ki67 low], luminal B (HER2-) (ER+ and/or PR+, HER2-, and Ki67 high), luminal B (HER2+) (ER+ and/or PR+ and HER2+), HER2 overexpressing (ER-, PR-, and HER2+), and triple negative (ER-, PR-, and HER2-). RESULTS: Screen-detected cancers had favorable clinicopathological characteristics, such as smaller tumor size and a lower frequency of lymph node involvement. Women with screen-detected cancers had a survival advantage. Subtype distribution differed significantly among women with screen-detected and symptomatic cancer. Screen-detected cancers were more likely to be luminal A and less likely to be HER2 overexpressing or triple negative cancer compared with symptomatic cancers (luminal A 61.3 vs. 44.2%, HER2 overexpressing 4.0 vs. 8.0%, triple negative 8.0 vs. 15.9%). Node status, mode of detection, and subtype were independent prognostic factors in the multivariate analysis. CONCLUSIONS: Differences in subtype distribution between screen-detected and symptomatic cancer could partially explain differences in outcomes.

Transcriptional regulation of the estrogen-inducible pS2 breast cancer marker gene by the ERR family of orphan nuclear receptors.

The estrogen-receptor-related receptors (ERRs) alpha, beta, and gamma are orphan nuclear hormone receptors that share significant homology with the estrogen receptors (ERs) but are not activated by natural estrogens. In contrast, the ERRs display constitutive transcriptional activity in the absence of exogenously added ligand. However, the ERRs bind to the estrogen response element and to the extended half-sites of which a subset can also be recognized by ERalpha, suggesting that ERRs and ERs may control overlapping regulatory pathways. To test this hypothesis, we explored the possibility that ERRs could regulate the expression of the estrogen-inducible pS2 gene, a human breast cancer prognostic marker. Transfection studies show that all of the ERR isoforms can activate the pS2 promoter in a variety of cell types, including breast cancer cell lines. Surprisingly, sequence analysis combined with mutational studies revealed that, in addition to the well-characterized estrogen response element, the presence of a functional extended half-site within the pS2 promoter is also required for complete response to both ER and ERR pathways. We show that ERR transcriptional activity on the pS2 promoter is considerably enhanced in the presence of all three members of the steroid receptor coactivator family but is completely abolished on treatment with the synthetic estrogen diethylstilbestrol, a recently described inhibitor of ERR function. Finally, we demonstrate that ERRalpha is the major isoform expressed in human breast cancer cell lines and that diethylstilbestrol can inhibit the growth of both ER-positive and -negative cell lines. Taken together, these results demonstrate that estrogen-inducible genes such as pS2 can be ERR targets and suggest that pharmacological modulation of ERRalpha activity may have therapeutic value in the treatment of breast cancer.

Changes in sensitivity to anticancer drugs during TPA-induced cellular differentiation in a human T-lymphoblastoid cell line (MOLT-4).

After four days of treatment with 10(-8) M TPA, differentiation of the human T-lymphoblastoid cell line MOLT-4 was induced along the T cell lineage, confirmed by a fall in adenosine deaminase and 5'-ectonucleotidase and a rise in purine nucleoside phosphorylase activity. TPA-treated cells became resistant to the cytotoxic effects of 1-beta-D-arabinofuranosylcytosine (Ara-C), 9-beta-D-arabinofuranosyladenine (Ara-A), and 2-chlorodeoxyadenosine. This was, in part, due to the altered cell cycle distribution (accumulation of cells in the G1 phase), since the toxicity of Ara-C and Ara-A is S phase specific. The diminished rate of Ara-C transport concomitant with Ara-CTP formation after TPA treatment is considered to be the biochemical basis for this acquired resistance.

Biochemical characterization of U937 cells resistant to L-asparaginase: the role of asparagine synthetase.

A human histiocytic lymphoma cell line, U937, is highly sensitive to L-asparaginase with an ID50 of about 0.0001 U/ml after 72 hr of culture. When U937 cells were made resistant to either L-asparaginase (1 U/ml) or asparagine deprivation, the activity of asparagine synthetase increased to 80- or 7-fold of the wild type, respectively. The phenotype of the resistance to L-asparaginase turned out to be stable under nonselective conditions for over several months. The hybrids between L-asparaginase sensitive (Molt4) and resistant (HL-60) cell lines revealed the latter phenotype in terms of L-asparaginase sensitivity and the activity of asparagine synthetase. Furthermore, U937 cells resistant to L-asparaginase could survive in glutamine-free media with 1.5-fold elevation of glutamine synthetase activity. These results altogether clarify the role of asparagine synthetase in L-asparaginase toxicity and have a good implication for the clinical use of L-asparaginase.

Calcitonin induces IL-6 production via both PKA and PKC pathways in the pituitary folliculo-stellate cell line.

It has been demonstrated that calcitonin-binding sites are present in a variety of tissue types, including in the pituitary gland. Interleukin-6 (IL-6) is also produced in the pituitary and it regulates the secretion of various hormones. In this study, we examined the expression of the calcitonin receptor and the mechanism of IL-6 production induced by calcitonin in the pituitary folliculo-stellate cell line (TtT/GF). The mRNA of calcitonin receptor subtype C1a, but not that of C1b, was detected by RT-PCR in TtT/GF cells and in the normal mouse pituitary. Calcitonin increased cAMP accumulation and IL-6 production in a concentration-dependent manner in TtT/GF cells. As calcitonin activates the PKA and PKC pathways, we investigated the contributions of PKA and PKC to IL-6 production. IL-6 production was only slightly increased by either 8-bromo-cAMP (1 mM) or phorbol 12-myristate 13-acetate (100 nM) alone. However, IL-6 was synergistically induced in the presence of both 8-bromo-cAMP (1 mM) and phorbol 12myristate 13-acetate (100 nM). Furthermore, calcitonin-induced IL-6 production was completely suppressed by H-89 (PKA inhibitor) or GF109203X (PKC inhibitor), indicating that the activation of both PKA and PKC is necessary for calcitonin-induced IL-6 production. On the other hand, pertussis toxin (G(i)/G(o) signaling inhibitor) treatment achieved an approximately 9-fold increase in calcitonin-induced IL-6 production. These results show that calcitonin-stimulated IL-6 production is mediated via both PKA- and PKC-signaling pathways, whereas calcitonin also suppresses IL-6 production by activating G(i)/G(o) proteins in folliculo-stellate cells.

Protein kinase A-dependent IL-6 production induced by calcitonin in human glioblastoma A172 cells.

In human glioblastoma A172 cells, interleukin-6 (IL-6) production was induced by interleukin-1 beta (IL-1 beta) and dibutyryl cyclic AMP. These cells have been shown to induce IL-6 production via a cAMP-protein kinase A system. Since calcitonin (CT) and calcitonin gene-related peptide (CGRP) are known to increase cAMP accumulation in murine and rat astrocytes, we examined whether these neuropeptides induced IL-6 production in A172 cells. Human CT and human CGRP increased IL-6 production and cAMP accumulation in a dose-dependent manner. A specific protein kinase A inhibitor, H-89, inhibited both CT- and CGRP-induced IL-6 production. CT and CGRP have been shown to cross-react with each other. To exclude the possibility of this cross-reactivity, we studied the additive effects of CT and CGRP and the inhibitory effects of specific inhibitors. When 100 nM CT was added, cAMP accumulation stimulated by 10 nM CGRP (the maximal dose) was increased. CGRP (8-37), a specific CGRP receptor inhibitor, inhibited cAMP accumulation and IL-6 production induced by CGRP, but did not inhibit these effects when they were induced by CT. Salmon CT (8-32), a specific inhibitor of the CT receptor, inhibited cAMP accumulation induced by CT, but did not inhibit the effect induced by CGRP. These results demonstrated that CT can induce IL-6 production via cAMP accumulation and the effects of CT are mediated via its own receptors.

Synergistic effect of methotrexate and 1-beta-D-arabinofuranosylcytosine on the generation of DNA strand breaks in a human promyelocytic leukemia cell line.

We investigated the accumulation of DNA strand breaks in a human promyelocytic leukemia cell line, HL-60, treated with methotrexate (MTX) and 1-beta-D-arabinofuranosylcytosine (Ara-C). The sequential treatment with MTX then Ara-C had a synergistic effect on the formation of DNA strand breaks, which was dependent on MTX and Ara-C concentrations. On the other hand, when Ara-C preceded MTX, no such synergism was observed. The addition of both thymidine and hypoxanthine to this system, but not thymidine or hypoxanthine alone, abolished the synergism. Pretreatment with MTX augmented the generation of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate. However, this augmentation did not necessarily correlate with the amount of strand breaks. Whatever the underlying mechanism of this synergism is, our present data provide one possible biochemical basis for sequential MTX and Ara-C therapy.

NG-nitro-L-[3H]arginine binding properties of neuronal nitric oxide synthase in rat brain.

NG-Nitro-L-arginine (L-NNA), a derivative of L-arginine (L-Arg), is known as a pseudosubstrate and inhibitor for nitric oxide synthase (NOS). To clarify the regulatory mechanism of substrate-binding domain in neuronal NOS (nNOS), we examined the characteristics of NG-nitro-L-[3H]Arg (L-[3H]NNA) binding using the cytosolic fraction and purified nNOS from the rat cerebellum, in comparison with L-[14C]citrulline formation from L-[14C]Arg. The L-[3H]NNA binding was inhibited by L-NNA > NG-methyl-L-Arg > diphenyleneiodonium > L-Arg, but was not inhibited by L-citrulline and D-Arg. Thus, L-NNA seems to bind the substrate-binding domain in the nNOS with high affinity rather than L-Arg. Even in the absence of NADPH, tetrahydrobiopterin (BH4) and Ca2+, the L-[3H]NNA binding activity was observed in the cerebellar cytosol, although L-[14C]citrulline could not be produced from L-[14C]Arg. L-[3H]NNA binding was increased by BH4 alone and was markedly enhanced by NADPH plus BH4 (NADPH/BH4), but not by Ca2+/CaM. In contrast, L-[14C]citrulline was formed only in the presence of NADPH/BH4 and Ca2+. Similar results were obtained in purified nNOS. These results suggest that L-[3H]NNA seems to bind the substrate-binding domain in the nNOS but the binding affinity of L-Arg was lower than the affinity of L-NNA. Although the substrate binding is necessary to BH4 and NADPH, Ca2+/CaM are further necessary for the formation of NO and L-citrulline.

Infection of the erythroid cell line, KU812Ep6 with human parvovirus B19 and its application to titration of B19 infectivity.

A human parvovirus B19 (B19) infectivity assay was developed using the erythroid cell line, KU812Ep6. KU812Ep6 was cloned for high efficiency infection with B19 in vitro, in the presence of erythropoietin by a limiting dilution method from the parent cell line, KU812. B19 was effectively propagated in KU812Ep6 and was detected for B19 antigens, VP1 and VP2. The titers of B19 positive sera measured with KU812Ep6 cells were in the range of 10(6) to 10(8) TCID50 ml. This KU812Ep6 infectivity assay had a 10(3)-10(4.5) higher sensitivity than the colony forming unit-erythroid (CFU-e) injury assay. It was calculated that one TCID50 needed 10(3) B19 genome copies, judging from the infectivity assay and semi-quantitative PCR. The KU812Ep6 infectivity assay was also used to determine infectivity of B19 in vitro, and to evaluate inactivation, as well as clearance of the virus. The inactivation of B19 by heating was carried out and infectivity declined from 10(4) TCID50 ml to < 10 TCID50 ml (lower limit of detection) at 60 degrees C for 3 h or at 70 degrees C for 30 min, but only decreased to 10(2.5) TCID50 ml at 50 degrees C for 8 h.

Effects of substance P and calcitonin gene-related peptide on axonal transport in isolated and cultured adult mouse dorsal root ganglion neurons.

Substance P and calcitonin gene-related peptide (CGRP) released from primary sensory neurons are known to play important roles in nociception and nociceptive transmission. In the present study, we attempted to clarify the roles of these neuropeptides in the regulation of axonal transport in sensory neurons. Cells were isolated from adult mouse dorsal root ganglia and cultured in F-12 medium containing fetal bovine serum for 48 h until their neurites were grown. These isolated and cultured DRG cells were mostly (>98%) small (diameter <25 microm) and medium (diameter, 25-40 microm) in size, and were immunoreactive for substance P and CGRP (85.9 and 66. 0% of total cells, respectively). Video-enhanced microscopy was applied to observe particles transported within neurites. Application of substance P (100 nM) decreased the number of particles transported in both anterograde and retrograde directions in each of DRG neurons tested (n=5). The instantaneous velocities of individual particles transported in anterograde and retrograde directions were also reduced by substance P. In contrast, alpha-CGRP (100 nM) increased the number of particles transported in both directions in each of DRG neurons tested (n=5), and also increased the instantaneous velocities of particles transported bidirectionally. Application of beta-CGRP (100-1000 nM) did not elicit any effect on axonal transport. Therefore, axonal transport in sensory neurons seems to be modulated by substance P and alpha-CGRP, both of which can be derived from its own and adjacent sensory neurons.

Chronic bullous disease with coexistent circulating IgG and IgA anti-basement membrane zone antibodies.

Coexistent circulating IgG and IgA anti-basement membrane zone (BMZ) antibodies were demonstrated in a patient with chronic bullous disease. Direct immunofluorescence microscopy showed IgG, IgA, and C3 deposits in a linear pattern at the BMZ in the patient's uninvolved skin. Ultrastructurally, the bulla was formed beneath the basal lamina as in dermatitis herpetiformis. This investigation might provide evidence for the immunologic overlap of bullous pemphigoid and dermatitis herpetiformis.

Prevention of 1-beta-D arabinofuranosylcytosine toxicity by 4-nitrobenzyl-6-thioinosine or dipyridamole in human leukemia cell lines.

The ability of the nucleoside transport inhibitors, 4-nitrobenzyl-6-thioinosine (NBTI) and dipyridamole (DP) to prevent 1-beta-D arabinofuranosylcytosine (Ara-C) toxicity was evaluated in two human leukemia cell lines, Molt 4 and HL-60. At non-toxic concentrations, DP (in Molt4 and HL-60) and NBTI (only in Molt 4) provided significant protection, whereas HL-60 was quite insensitive to NBTI. The different response of these two cell lines to NBTI and DP was also noted in the toxicity of other nucleoside analogs, including 9-beta-D arabinofuranosyladenine (Ara-A), 2'-chlorodeoxyadenosine (CdA), tubercidin and 5'-bromodeoxyuridne (BUdR). A transport study of [3H]-Ara-C revealed that NBTI partially inhibited the drug entry into HL-60 cells, which correlated well with Ara-CTP generation.

[Clinical evaluation of cefuzonam in pediatrics].

Cefuzonam (L-105, CZON), a new injectable cephalosporin, was used in 12 pediatric patients with infections. The following is a summary of the results: The 12 cases included 3 cases of tonsillitis (pathogen: Haemophilus parainfluenzae in 1 case, Haemophilus influenzae in 2 cases), 4 cases of pneumonia (Staphylococcus aureus in 1 case, pathogen unknown in 3 cases), 2 cases of nephropyelitis (Escherichia coli in 2 cases), 1 case of purulent lymphadenitis (pathogen unknown), 1 case of purulent thyroiditis (mixed infection of Streptococcus milleri, Haemophilus aphrophilus and anaerobes), and 1 case of vulvar abscess (E. coli). Dose levels of CZON were 42.9 approximately 93.3 mg/kg/day divided into 3 or 4 times and the drug was intravenously injected for 6 to 12 days. Clinical efficacies were excellent in 4 cases, good in 5 cases, and poor in 3 cases, with the efficacy rate of 75.0%. The 3 cases with poor efficacy consisted of 1 case each of pneumonia complicated with chronic granulomatosis, purulent thyroiditis associated with piriform recess fistula, and purulent lymphadenitis of armpit developed after surgical operation of congenital heart disease. In the first 2 cases satisfactory efficacy was not obtained by chemotherapy alone, and complete cure was seen after surgical operation. Side effects were not observed clinically. One case each of slight prolongation of prothrombin time and transient elevations of GOT and GPT values were noted but no severe abnormalities were found in laboratory tests.(ABSTRACT TRUNCATED AT 250 WORDS)

[Allogeneic bone marrow transplantation in a case of acute lymphoblastic leukemia with positive Philadelphia chromosome].

A 12-year-old boy with Philadelphia chromosome positive acute lymphoblastic leukemia received bone marrow transplantation (BMT) from an HLA identical sibling during the second remission. The diagnosis was made at the age of nine. Laboratory examination on admission revealed remarkable leukocytosis (92,000/microliters) with 93% lymphoblasts in the peripheral blood. Blastic cells were FAB L1 common ALL. Chromosomal study on both peripheral blood and bone marrow cells showed that lymphoblasts had an abnormal karyotype of 47, XY, inv (9), t(9; 22), +17. One month later he achieved remission by induction therapy consisting of vincristine, L-asparaginase, doxorubicin, and prednisolone. He was given intrathecal injection of methotrexate and cranial irradiation of 24 Gy for CNS prophylaxis. The cells with Philadelphia chromosome disappeared during remission. Hematological relapse occurred twenty one months later after first remission on April, 1986. He received re-induction therapy including L-Asp VDP, and high-doses of cyclophosphamide, methotrexate and araC. He obtained karyotypic remission on October 1986. Subsequently, bone marrow transplantation was performed following high-dose araC, CY and TBI as preconditioning on December 18, 1986. Methotrexate and cyclosporin A were given intravenously to prevent GVHD. On day 14, karyotypic conversion was detected, suggesting the successful bone marrow grafting. Acute GVHD appeared on day 25, and was treated with prednisolone and cyclosporin A. Prednisolone was tapered by day 80. On day 91, cyclosporin A was discontinued because herpes zoster occurred. Acyclovir was effective, but skin GVHD reappeared. With low-dose prednisolone, skin GVHD improved. Sicca syndrome soon appeared and was followed by chronic GVHD.(ABSTRACT TRUNCATED AT 250 WORDS)

Total synthesis of ardisiaquinone A, a potent 5-lipoxygenase inhibitor, isolated from Ardisia sieboldii, and degree of 5-lipoxygenase inhibitory activity of its derivatives.

Ardisiaquinone A (1), isolated as a potent 5-lipoxygenase inhibitor from the woods of Ardisia sieboldii, has been synthesized efficiently via a cross-coupling reaction between the yne 5 and the iodide 6 derived from the common intermediate 4. Inhibitory activity for 1 and its derivatives is also reported.

Naturally occurring 5-lipoxygenase inhibitors. VI. Structures of ardisiaquinones D, E, and F from Ardisia sieboldii.

New 1,4-benzoquinone derivatives, ardisiaquinones D(2), E(4), and F(5) along with the known ardisiaquinones A(1) and B(3) have been isolated from the leaves of Ardisia sieboldii (Myrsinaceae) and shown to be 5-lipoxygenase inhibitors. Their structures have been elucidated by spectroscopic analysis and chemical degradation. The degree of inhibition of 5-lipoxygenase activity by the ardisiaquinones and some derivatives of ardisiaquinone A is reported.